Mothers against dpp
The role of Mad in Dpp-mediated signaling was examined by utilizing tkvQ199D, an activated form of the Dpp type I receptor serine-threonine kinase thick veins (tkv). In the midgut, dpp is expressed in the visceral mesoderm of parasegments 3 and 7. In response to Dpp signals, cells expressing dpp in parasegment 3 repress the expression of the homeotic gene Sex combs reduced. Dpp signals are also required to maintain dpp expression in parasegment 3 through an autocrine feedback loop. However, cells in parasegment 4 do not appear to be affected by Dpp signals; Scr is expressed while dpp is not. In ps7, the homeotic gene Ultrabithorax initiates dpp expression. Subsequently, Dpp functions in an autocrine manner to maintain Ubx as well as its own. In ps7, Dpp also signals between germ layers to the underlying endoderm. Within the midgut endoderm, which does not express dpp, expression of the homeotic gene labial is dependent on Dpp signals (Newfeld, 1997).
In the embryonic midgut, tkvQ199D mimics Dpp-mediated inductive interactions. There is an anterior expansion of labial midgut endoderm expression in response to ubiquitously expressed tkvQ199D. In early stage tkvQ199D embryos, dpp expressin is expanded to include ps4, ps5 and ps6. In late stage tkvQ199D embryos, the expanded domain of expression is maintained at very high levels, while in late stage Mad mull tkvQ199D embryos, this is not observed. Analysis of Scr expression in Mad null embryos, combined with tkvQ199D, reveals an anterior expansion of Scr, showing that Mad and dpp are required for repressing Scr. Mad function is epistatic to tkvQ199D in the repression of Scr. Thus homozygous Mad mutations block signaling by tkvQ199D and appropriate responses to signaling by tkvQ199D are restored
by expression of MAD protein in Dpp-target cells (Newfeld, 1997).
Endogenous Mad is
phosphorylated in a ligand-dependent manner in Drosophila cell culture. Dpp
overexpression in the embryonic midgut induces Mad nuclear accumulation; after
withdrawal of the overexpressed Dpp signal, Mad is detected only in the cytoplasm.
However, in three different tissues and developmental stages actively responding to
endogenous Dpp, Mad protein is detected in the cytoplasm but not in the nucleus. To date, in live animals, the nuclear accumulation of Mad in response to Dpp has been observed only with Dpp overexpression. It is possible that even under conditions of maximal endogenous signaling the fraction of Mad translocated to the nucleus is too small to be detected by current techniques. Alternatively Mad's biological role is independent of observed subcellular translocation (Newfeld, 1997).
The function of the Drosophila mef2 gene, a member of the MADS box supergene family of
transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely
somatic, visceral, and cardiac. During embryogenesis, mef2 undergoes multiple phases of expression,
which are characterized by initial broad mesodermal expression, followed by restricted expression in
the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the
differentiated musculatures. Evidence is presented that temporally and spatially specific
mef2 expression is controlled by a complex array of cis-acting regulatory modules that are responsive
to different genetic signals. Functional testing of approximately 12 kb of 5' flanking region of the mef2
gene shows that the initial widespread mesodermal expression is achieved through a 280-bp
twist-dependent enhancer. Subsequent dorsal mesoderm-restricted mef2 expression is mediated
through a 460-bp dpp-responsive regulatory module, which involves the function of the Smad4 homolog
Medea and contains several binding sites for Medea and Mad. Regulated
mef2 expression in the caudal and trunk visceral mesoderm, which give rise to longitudinal and circular
gut musculatures, respectively, is under the control of distinct enhancer elements. In addition, mef2
expression in the cardioblasts of the heart is dependent on at least two distinct enhancers, which are
active at different periods during embryogenesis. Mef2 expressing cells are coincident with those expressing Tinman. Notably, both Mef2 and Tinman expression are in four of six cardioblasts that are present per hemisegment. The complete overlap between the two expression patterns suggests that the activity of this enhancer element could be dependent on tinman function or under similar regulatory controls as is tinman. The cardiac enhancer that functions at later stages also drives mef2 expression in the caudal visceral mesoderm as well as in the somatic mesoderm. Moreover, multiple regulatory elements are
differentially activated for specific expression in presumptive muscle founders, prefusion myoblasts,
and differentiated muscle fibers. Taken together, the presented data suggest that specific expression of
the mef2 gene in myogenic lineages in the Drosophila embryo is the result of multiple genetic inputs
that act in an additive manner on distinct enhancers in the 5' flanking region (Nguyen, 1999).
spalt is a target of MAD in the wing. Overexpression of dpp causes an expansion of the wing along the AP axis, the width of the spalt domain expanding relative to the DPP stripe. Cells with reduced MAD fail to express spalt, suggesting that cells must be able to transduce the DPP signal to express spalt (Lecuit, 1996).
Loss of DPP signaling in the Drosophila eye can lead to ectopic expression of wingless, suggesting that MAD negatively regulates wingless transcription. Mutant Mad clones are found to express wingless in eye imaginal discs. Similarly, bifurcations of antennae are associated with mutant Mad clones. Such clones express wg and are overgrown when located in the dorsal region of the antennal disc. Thus the antagonistic effect of DPP signaling in wg expression is also observed in other discs and might be a general mechanism during Drosophila imaginal disc development (Wiersdorff, 1996).
The observation that dpp is not expressed in mutant Mad eye margin clones raises the possibility that DPP signaling is required for the maintenance of dpp expression. Supporting this, mutation in dpp itself reduces expression of a dpp reporter gene in the eye margin (Wiersdorff, 1996).
The spalt and optomotor-blind genes are expressed in a nested pattern in the wing imaginal disc, centered on and extending up to 20 cell diameters away from the stripe of decapentaplegic expression along the anteroposterior compartment boundary. The vestigial gene is even more broadly expressed and is required in all cells of the developing wing. Activation of vg through the quadrant enhancer is responsible for the broad expression of vg in the wing blade. The quadrant enhancer is found in an 806 bp DNA segment in the fourth intron of the vg gene. This enhancer is sufficient to drive expression in all of the developing wing blade. vg expression in developing wing-blade cells, beginning from the wing-blade primordium of the wing imaginal disc, requires a dpp signal from the A/P boundary and another signal from the D/V boundary. The term quadrant enhancer refers to the expression pattern, which is bisected by non-expressing DV boundary cells and becomes less intense along the A/P boundary in the late third instar, producing a quadrant-like pattern. Unlike the D/V boundary enhancer, which is regulated by the Notch pathway and is active in the second instar, the quadrant enhancer is not active until the early third instar. The quadrant enhancer is Suppressor of hairless independent, suggesting that Notch function is not required for formation of this part of the wing during the third instar. The temporal order of D/V boundary and quadrant enhancer activation suggests that D/V boundary cells must produce a signal (or signals) that organize the rest of the wing, including expression from the quadrant enhancer. Activation of the quadrant enhancer requires dpp, since thick veins mutants exhibit reduced Vg protein levels and have smaller wings and wing discs (Kim, 1996).
Because Mad is an intracellular signal transducer of Dpp receptors, the requirement of Mad activity for Vg expression in the wing imaginal disc was examined in mitotic clones with reduced Mad function. Mad has no effect on the Notch-dependent dorsoventral boundary enhancer, but the quadrant enhancer requires Dpp signaling and Mad function. The N-terminal MAD homology region 1 (MH1) plus the central less conserved proline-rich linker region bind DNA and protect a single interval within the quadrant enhancer. A 39 bp double stranded oligonucleotide of the vg quadrant enhancer contains the Mad-protected region. Mutating 12 base pairs within this region prevents Mad-directed expression. The C-terminal MH2 domain of Mad effectively inhibits DNA binding and suggests a mechanism that might contribute to inactivation of Mad in the absence of Dpp signaling. Examination of the Ultrabithorax midgut enhancer, for which the Dpp response element has been localized to the 95 bp DI-DII interval, and the labial endoderm enhancer reveals a Mad binding-site consensus of GCCGnCGC. The two sites of highest affinity match this consensus perfectly, and three lower-affinity sites contain mismatches in one or as many as three positions (Kim, 1997).
In both Drosophila and Xenopus embryos, gradients of Dpp/BMP activity are established that are responsible for patterning along the dorsoventral axis. Dpp activity has its highest
levels along the dorsal midline of the cellular blastoderm embryo and declines toward more lateral regions where it is inhibited by the product of the short gastrulation (sog) gene. The high levels determine the cell fate of the amnioserosa
in the dorsal-most cells, whereas lower levels specify aspects of the dorsal epidermis in dorsolateral cells. The absence of Dpp activity in ventrolateral regions permits
the formation of the neurogenic ectoderm, which gives rise to both the ventral epidermis and the central nervous system (Rushlow, 2001).
How does Dpp specify cell fate in a concentration-dependent manner? It is thought that Dpp signaling in the early embryo regulates the transcription of downstream
target genes that are expressed in nested domains centered around the dorsal midline. High-level Dpp targets such as Race and u-shaped (ush) are expressed in the presumptive amnioserosa. pannier
(pnr) is expressed in a broader domain that spans the amnioserosa and part of the dorsal ectoderm. Thus, it requires lower levels of Dpp.
Finally, low-level targets such as early zerknullt (zen) and dpp are expressed in an even broader domain that abuts the ventral ectoderm. A possible molecular mechanism to explain the threshold responses of Dpp target genes is that their promoters have different affinities to
Smads and therefore can be induced by different levels of nuclear Smads, similar to the mechanism of differential activation by the Drosophila morphogens Dorsal
(Dl) and Bicoid (Bcd). The fact that an additional mechanism is involved came
from the characterization of the brinker (brk) gene. brk
negatively regulates low-level and intermediate-level target genes. Study of the response elements of these target genes can therefore provide clues about the
mechanisms of threshold responses to the Dpp morphogen, as well as the interplay of positive and negative inputs in the expression of target genes (Rushlow, 2001 and references therein).
zen has a dynamic pattern of expression in the early embryo. During precellular nuclear division cycles 11-13
and during early cellularization (nuclear cycle 14), zen is expressed in a broad dorsal-on/ventral-off pattern. This pattern is thought to be activated by an unknown
ubiquitous activator present throughout the embryo and repressed by the Dl morphogen localized in ventral regions. It is
Dpp-independent because early zen expression is normal in dpp null mutants. However, slightly later,
during early to mid-cellularization, maintenance of the zen pattern becomes dependent on Dpp because zen transcripts fade away suddenly in dpp null mutants. It also becomes dependent on Brk repression because zen transcripts expand into the ventral ectoderm
in brk mutants. Thus, the broad pattern of zen is maintained by Dpp in the dorsal region and repressed by Brk in ventral regions.
During mid- to late-cellularization, this pattern undergoes a process of refinement in which zen transcripts are lost from the lateral regions and become restricted to a
narrow domain of the dorsal-most cells. Brk plays no role in refinement because in brk mutants, although zen expands ventrally, it refines normally (Rushlow, 2001 and references therein).
zen expression is directed by 1.6 kb of 5' flanking DNA sequences referred to as the zen promoter. The distal part of the promoter
between 1.2 and 1.4 kb is responsible for Dl-dependent ventral repression. Sequences
required for the initiation, maintenance, and refined expression of zen are located in the proximal 0.7 kb of the promoter, but they are not well-characterized (Rushlow, 2001 and references therein).
The regulation of zen during cellular blastoderm formation has been analyzed. Low levels of the Dpp signal transducer
p-Mad (phosphorylated Mad), together with Brinker, define the spatial limits of zen transcription in a broad dorsal-on/ventral-off domain. The subsequent refinement of this pattern to the dorsal-most cells, however,
correlates with high levels of p-Mad that accumulate in the same region during late blastoderm. Examination of the zen regulatory sequences reveals the presence of multiple Mad and Brk binding sites, and these results indicate that a full occupancy of the Mad sites due to high concentrations of nuclear Mad is the primary
mechanism for refinement of zen. Interestingly, several Mad and Brk binding sites overlap, and it has been shown that Mad and Brk cannot bind simultaneously to such sites. A model is proposed whereby competition between Mad and Brk determines spatially restricted domains of expression of Dpp target genes (Rushlow, 2001).
Examination of p-Mad staining in wild-type embryos indicates that maintenance and refinement require different levels of signaling. Only the highest p-Mad levels
in the dorsal-most five to six nuclei are capable of driving zen transcription during late cellularization. The lower levels present in the three to four lateral nuclei to either side are not sufficient to activate zen, although earlier they were sufficient for its maintenance. This indicates that maintenance may
involve the contribution of an additional activator, perhaps the same ubiquitous activator that initiates zen earlier (Rushlow, 2001).
Later during refinement, p-Mad at peak levels is sufficient to up-regulate zen. Interestingly, the Dpp target gene ush is expressed in a broader domain than refined zen that includes the three to four lateral nuclei. This indicates that ush can be activated by a lower level of signaling than refined zen and that the high-level class of Dpp target genes can be further subdivided (Rushlow, 2001).
Because the amount of p-Mad depends on the amount of Dpp activity, the simplest explanation is that the zen promoter responds to differences in Dpp activity by measuring the level of nuclear Smads. Such a conclusion is consistent with the presence of multiple Mad/Medea binding sites and mutagenesis analysis. Deletion of only two Mad/Medea sites results in the loss of refined expression; therefore, most if not all of the Smad binding sites are required for this function, as are the peak levels of p-Mad activity (because weak dpp mutants do not refine). However,
maintenance is not affected, possibly because several sites remain intact and this function does not require full p-Mad activity. That the zen promoter measures the level of nuclear Smads also explains the broad dorsolateral pattern of both p-Mad immunostaining and zen expression in sog embryos. In the absence of
inhibition by Sog, Dpp continues to signal, and p-Mad can accumulate in the dorsolateral region of the embryo and induce zen expression (Rushlow, 2001).
The experiments presented here show that Mad/Medea and Brk regulate zen by binding to separated and overlapping DNA binding sites. There are 10 Mad/Medea
and 6 Brk binding sites in the zen promoter, 5 of which are shared, indicating duality in their function. Indeed, the results from mutagenesis of the zen promoter show that the shared sites mediate both Brk and Mad/Medea functions.
Five Brk and nine Smad binding sites are clustered in the zen proximal regulatory element over about 600 bp with spacing not exceeding 120 bp. This organization is similar to that of several well-studied enhancers from Drosophila. These enhancers are activated by a variety of transcriptional activators and repressed by short-range repressors such as Snail (Sna), Knirps (Kni), and Krüppel (Kr). All three of these repressors are DNA-binding proteins that can inhibit activator function when they
are bound not further than 150 bp away from the activator binding site. It has been shown that they all contain a short stretch of amino acids, P-DLS-K, that is required
for recruitment of the corepressor dCtBP. Analysis of zen regulation indicates that Brk also may be a short-range repressor. It is a
DNA-binding protein and contains a PMDLSG domain. Preliminary in vitro experiments showed that Brk interacts with dCtBP; however, embryos devoid of dCtBP activity do not ectopically express zen and dpp, indicating that dCtBP is dispensable for Brk repression and other corepressors interact with Brk, or that Brk repression of these targets does not require additional factors (Rushlow, 2001).
What remains illusive is the identity of the ubiquitous transcriptional activator that activates zen in the dorsal ectoderm during precellular stages and early cellularization. It is possible that this activator interacts with Smads to enhance transcription of zen at a time when p-Mad levels are low. Also, Brk represses the ubiquitous activator, because zen becomes ectopically expressed in brk mutants. Thus far, deletion analysis of the zen promoter has not uncovered any sequences that might interact with this putative activator. It is possible that these sequences are redundant and scattered over the entire promoter and may in fact overlap with Smad and/or Brk binding sites (Rushlow, 2001).
In the cellularizing embryo, Dpp and Brk activities overlap in the lateral-most region. Here Dpp and Brk function to set thresholds of response for target genes such
as zen and pnr. In this same region, Dpp signaling negatively regulates brk expression. Similarly, in the wing disc, the Brk expression
domain overlaps with that of the Dpp target gene omb in the region where activated p-Mad is present. It has been proposed that a dual mechanism whereby Dpp can simultaneously down-regulate Brk repressor levels and antagonize its function on target gene promoters would be very efficient in establishing sharp threshold responses. Based on the experiments described here, a molecular model is proposed to explain mechanistically the antagonizing activities of Brk and Smads. It is proposed that they are involved in direct competition for binding to shared binding sites on target
promoters. Thus, it is the balance of their opposing activities that determines the transcriptional state of the target genes. Two sets of experiments support this model:
(1) ectopic expression of Brk in eve-stripe 2 abolishes zen expression in those cells. The elevated level of Brk in the stripe was therefore sufficient to repress the zen promoter even in the presence of activated Smads. The possibility that zen is
repressed indirectly through Brk-mediated repression of dpp is highly unlikely because there was no delay in zen repression. (2) In vitro competition
experiments also support the model. Especially revealing is the fact that the outcome of competition depends on the relative concentrations of both proteins and their
binding affinities. Competitive mechanisms have been proposed to operate on many promoters where mutually exclusive DNA-binding
factors are involved, and, in some instances, DNA-binding assays similar to the ones used in this study were used to show competition for binding between activator and repressor proteins. For example, bHLH proteins compete with a zinc-finger
repressor for E-box binding in the immunoglobulin heavy chain enhancer (Rushlow, 2001 and references therein).
The findings presented here provide a framework for further study of the mechanisms of regulation of Dpp morphogen targets. zen is the only one of the known Dpp
target genes that responds to two threshold activities: low (during early to mid-cellularization) and high (during late cellularization). Based on the results presented here and the
proposed competition mechanism for activation and repression of the zen promoter, predictions can be made about the organization of the regulatory elements of the
other Dpp target genes. High-level targets such as ush strongly depend on high levels of Smads, and their regulatory elements may have many, and possibly closely
packed, Smad binding sites. Low-level targets such as omb in the wing imaginal disc may be repressed by Brk binding to their regulatory sequences. The spatial domains of expression of the intermediate targets such as pnr in the embryo and sal in the wing disc, which are dependent on both Dpp signaling and Brk repression, might be determined by the
net balance of positive and negative inputs. Interestingly, this type of mechanism can result in expression domains that vary largely in size and may result in even
broader domains than the low-level targets. An example is the vg gene. In third-instar imaginal wing discs, vg is expressed in a broader domain than omb. Its expression along the anterior-posterior boundary in the wing pouch is activated by the quadrant enhancer that contains Mad binding
sites essential for activation. At the same time, vg is repressed by Brk. However, the essential Smad binding sites do not match the Brk binding sites, like many of the Smad sites in the zen promoter, suggesting that they will have no or low
affinity for the Brk protein. Neither are there strong zen-like Brk binding sites in the quadrant enhancer. Its broad expression
domain could then be explained if the positive inputs from Smads, enhanced by signals from the dorsoventral boundary, are able to
overcome Brk repression far from the Dpp source (Rushlow, 2001).
Further studies of the arrangement, affinities, and numbers of repressor and activator sites in Dpp target promoters will determine to what extent the different
thresholds of responses to the Dpp morphogen activity are shaped by a simple balance of positive and negative transcriptional inputs (Rushlow, 2001).
Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study it is shown that Shn acts as a DNA-binding Mad cofactor in the nuclear response
to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the
regulation of endogenous Dpp target genes a cell culture assay was developed and it has been shown that Shn and Mad act synergistically to induce transcription. These results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor (Dai, 2000).
Although mutations in the binding sites for Mad result in
more severe loss of Ubx B expression compared to that
caused by mutations in the Shn sites, in neither case is the
expression abolished, raising the possibility that inputs
from both proteins contribute to the regulation of Ubx B.
There is increasing evidence that protein-protein interactions
between Smads and accessory transcriptional factors
can result in cooperative binding and synergistic transcription of reporter genes. The fact that a Ubx B reporter that
lacks Shn-binding sites (BS1S2) shows residual staining,
while Ubx B expression is completely absent in shn mutant
embryos, suggests that loss of Shn protein has more severe consequences than loss of
Shn-binding sites. In order to determine whether protein-protein
interactions as well as DNA-binding contribute to
activation of Ubx B by Shn and Mad, an assay
was developed to study the nuclear response to Dpp signaling.
The B enhancer was cloned upstream of a minimal promoter
driving expression of the luciferase gene (Ubx B-Luc)
and its activity was examined in cultured cells. This reporter shows very low levels of basal expression in the BMP-responsive C3H10T1/2 cells. Cotransfection with Mad/
Medea results in only a slight elevation of luciferase
activity. However coexpression of Mad/Medea with constitutively
activated TkvA results in a dramatic 25-fold
increase in promoter activity relative to the basal response.
In other words, coexpression of all three components
causes a 5-fold stronger stimulation than expression of
either Mad/Medea or TkvA alone. The response to TkvA
is dependent on Mad and Medea since transfection with
the receptor alone leads to only a small increase in transcription
over basal levels, perhaps due to phosphorylation of
endogenous BMP-specific Smads (Dai, 2000).
Whether coexpression of Shn with Mad and Medea could enhance transcriptional
activation of Ubx B-Luc was examined. Expression of Shn or Mad/Medea alone elicits a weak transcriptional response. However coexpression of all three
proteins results in a 32-fold induction of reporter gene
activity relative to the basal response. This is a
6-fold increase over the response to either Shn or Mad/Medea alone. More strikingly this induction is 3-fold
greater than the expected additive response to expression of
the individual proteins. To test the importance of Mad and
Shn DNA-binding to synergistic activation, a luciferase reporter construct was generated lacking both Mad
sites known to be required for expression in the embryo
(Ubx BM2). As anticipated, it was found
that the response of Ubx BM2-Luc to stimulation by TkvA
and Mad is significantly reduced when compared to
wild-type Ubx B. Interestingly, however, deletion
of the Mad-binding sites in BM2 does not affect the induction
of reporter activity by Mad/Medea in the presence of Shn. In analogous experiments using a Ubx BS1S2-Luc
reporter, loss of the Shn-binding sites only marginally
affects the cooperative response to Shn and Mad/Medea. These results could indicate that synergistic transcriptional activation by overexpression of Shn and
Mad/Medea does not depend entirely on their ability to
bind DNA, but involves cooperative protein-protein interactions (Dai, 2000).
To test this, a reporter was constructed that lacks
both Mad as well as Shn-binding sites (Ubx BM2S1S2-luc).
The response of Ubx B to overexpression
of Mad/Medea and Shn is strongly reduced in the double
mutant. It is concluded that binding sites for either Mad or
Shn are sufficient to mediate synergistic activation of the
Ubx B reporter. However, when neither protein can bind the
enhancer, it is no longer possible to elicit a transcriptional
response. While the data may be interpreted as
redundancy for Mad/Medea and Shn in stimulating UbxB
transcription, this view is contradicted by the fact that
expression of either protein alone clearly does not stimulate
maximal response of the UbxB reporter. Taken
together these data indicate that Shn can act as a transcriptional
coactivator with Mad to regulate the expression of
the Ubx B enhancer (Dai, 2000).
Signaling by Dpp activates targets such as vestigial indirectly through negative regulation of brinker. The Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity is found in an amino-terminal region containing a putative homeodomain. Brk binds to a Dpp
response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for Mad. Furthermore, Brk is able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs from the Smad3/Smad4 binding site consensus. The Dpp response element from Ubx is more sensitive to repression by Brk than is the vg quadrant enhancer. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Evidence suggests that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad (Kirkpatrick, 2001).
Binding of Brk to the Ubx and vg probes generates multiple bands, possibly indicating that Brk binds to more than one site. The Ubx element contains an inverted repeat of GGCGCT that overlaps a
previously identified Mad binding site. Whereas the Mad site embedded in this repeat resembles the vg Mad site, the repeat as a whole is only matched at
7 of 12 positions in vg. Brk was tested for the ability to bind one copy of this sequence in a DNA probe that was otherwise divergent in sequence from the
Ubx element. Brk binds to the GGCGCT probe with affinity that is similar to its affinity for the Ubx probe and
yields a single major shifted band at about the same position as the lower most band observed with the Ubx probe. Although two weak upper bands are also
observed with the GGCGCT probe, overall, these results are consistent with high affinity interaction of Brk with just one site in the GGCGCT probe (Kirkpatrick, 2001).
To investigate the specificity of Brk for the GGCGCT sequence, the effects of single base pair substitutions were determined. This was done measuring the ability of
unlabeled 'wildtype' (GGCGCT) and mutant DNAs to compete with the labeled GGCGCT probe. In all, five mutants exhibited an ~20-fold reduction in the
binding affinity, whereas the least critical position contributed as much as 3-fold to binding affinity. These results indicate that Brk makes base-specific contacts across
the entire GGCGCT sequence (Kirkpatrick, 2001).
The GGCGCT repeat in the Ubx element overlaps a Mad binding site that can be modeled to consist of two degenerate Smad boxes, suggesting that
Brk may compete with Mad for binding. This could not be determined unequivocally using the Ubx probe because Mad and Brk complexes have nearly
identical mobilities in the band shift assay. However, the GGCGCT probe forms a complex with Mad that is easily resolved from the main complex formed
with Brk; this probe makes clear that formation of Brk complexes correlates with reduced binding of Mad. In contrast, the same
amount of Brk did not reduce binding of Mad to the M7 probe, evidence that Brk reduces the level of Mad binding by competition
rather than by sequence-independent inhibition (Kirkpatrick, 2001).
To determine whether the Brk binding sites identified using the band shift assay are actually required for repression, the Ubx element was mutated to disrupt Brk binding. Each of three GGCG(C/T) sequences was changed to GTCG or to GGCGA, both of which dramatically reduce Brk binding but still allow Mad to bind. Introduction of the same triple-substitutions into the
2×Ubx-lacZ reporter result in an ~100-fold decrease in sensitivity to repression by cotransfected Brk. These results demonstrate that Brk binding sites are required for repression and confirm that the sequence specificity characterized in band shift experiments is also observed in cells (Kirkpatrick, 2001).
The overlap of Mad and Brk binding sites in the Ubx midgut element suggests that Brk might repress Dpp targets by
simply competing with Mad for occupancy of an enhancer element. However, repressors generally function by quenching the activating potential of transcription
factors bound nearby or by means of long range interfering effects on the general transcription machinery. To determine whether Brk is capable of functioning as
an active repressor, Brk binding sites were positioned adjacent to sites for the unrelated Notch-responsive activator, Suppressor of Hairless [Su(H)] and
reporter expression was monitored in response to cotransfected Brk, Su(H), and activated Notch effector plasmids. Brk completely prevents activation by Su(H), whereas
a control reporter containing only Su(H) sites was repressed only 2-4-fold, an effect that may have been caused by the presence of a single Brk binding site adjacent
to the hsp70 TATA box. Given this ability of Brk to function as a generic active repressor, it is reasonable to speculate that Brk might control a subset of Dpp targets
without the direct involvement of Mad (Kirkpatrick, 2001).
Brinker is a nuclear protein that antagonizes Dpp signalling in Drosophila. Ultrabithorax (Ubx) is a HOX gene that activates, and responds to, the localized expression of Dpp during endoderm induction. Ubx expression is negatively regulated by Dpp. Brinker represses Ubx in the embryonic midgut. The functional target for Brinker repression coincides with the Dpp response sequence in the Ubx midgut enhancer, namely a tandem of binding sites for the Dpp effector Mad. Brinker efficiently competes with Mad in vitro, preventing the latter from binding to these sites. Brinker also competes with activated Mad in vivo, blocking the stimulation of the Ubx enhancer in response to simultaneous Dpp signalling. These results indicate how Brinker acts as a dominant repressor of Dpp target genes, and explain why Brinker is a potent antagonist of Dpp (Saller, 2001).
The control of Ubx by Dpp and Wg signalling has been studied by functional dissection of a minimal midgut enhancer called Ubx B. This enhancer directs expression of a linked ß-galactosidase (lacZ) gene in parasegments (ps) 6-9, and also in ps3, of the midgut mesoderm as a result of stimulation by Dpp and Wg, which are expressed in or near these regions. This stimulation requires distinct Dpp and Wg response sequences (DRS and WRS) within Ubx B. In addition, Ubx B is repressible by high Wg levels near the Wg signalling source, and is also repressed in the absence of Wg signalling in cells remote from the source. The former repression is mediated by the WRS-R, a sequence coinciding with the Mad binding sites within the DRS, the latter by the WRS, a binding site for Pangolin, the Drosophila T cell factor (Saller, 2001).
Since Ubx is a Dpp target gene in the embryonic midgut, it was asked whether this HOX gene might be under brk control. Thus, brk mutant embryos were stained with an antibody against Ubx and weak ectopic Ubx staining was found in the posterior midgut mesoderm of these mutants. Normally, the HOX protein Abdominal-A represses Ubx in the posterior midgut, but evidently this is not sufficient to keep Ubx repressed in the absence of brk. However, no Ubx derepression was observed in the anterior midgut of brk mutants, probably because of the silencing of Ubx in this region by Polycomb. But derepression was found in the anterior and posterior midgut of brk mutant embryos when examining lacZ expression conferred by an extensive Ubx midgut enhancer called RP9, the expression of which closely resembles Ubx expression in the midgut. These stainings show that brk represses Ubx in the embryonic midgut (Saller, 2001).
Next, a series of mutant versions of Ubx B was tested that carry nested point mutations. Most of these are still derepressed in brk mutants, e.g. BM1, which has a mutated MadB site. However, three mutant enhancers were no longer derepressed: B4, which has a mutated Pangolin binding site; B4R8, which carries a mutation in a conserved sequence motif; and BM2, in which both Mad binding sites are mutated. Formally, each of these mutations could define a target for Brinker repression. Alternatively, they define sequences that are essential for enhancer activation, in particular for ectopic activation at the midgut ends. This is a clear possibility since B4, BM2 and B4R8 are each considerably less active than Ubx B and other mutant enhancers such as BM1 (Saller, 2001).
Full-length and various fragments of Brinker were expressed as glutathione S-transferase (GST) fusion proteins in bacteria, in order to test whether these fusion proteins can bind to the signal-responsive sequence from Ubx B in gel shift assays. This revealed that full-length Brinker, or its N-terminus alone, can bind to this sequence, whereas the C-terminus cannot. This is consistent with the suggestion that the N-terminus contains a putative DNA binding domain similar to the homeodomain. Indeed, a minimal fragment spanning this domain (BRK44-99) binds to the probe as well as full-length Brinker (Saller, 2001).
Next, Brinker binding to mutant DNA probes was tested. Of these, BM2 and BM0 are the only mutants that no longer show any binding to Brinker. Likewise, Brinker binding to DNA can be competed with an excess of unlabelled wild-type probe, but not with mutant BM2 probe. This shows that Brinker binds to Ubx B in a sequence-specific manner, and that the residues mutated in BM2 and BM0 are critical for Brinker binding (Saller, 2001).
Three perfect matches to a consensus site for Brinker binding, GGCG C/T C/T, are found in Ubx B. These are adjacent to one another, and each of them is mutated in BM2. The results with BM1 indicate that the first of these matches (Brk bs1) is sufficient for Brinker function in vivo and in vitro. However, Brk bs3 alone is unlikely to be sufficient for function, given that Brinker cannot bind to the mutant probe BM0. Finally, the results indicate that Brk bs2 (perhaps together with bs3) can substitute for Brk bs1 and provide full function: BC2 is repressible by brk in vivo, and Brinker binds to BC2, BC and BM01 mutant probes, all of which lack Brk bs1 (Saller, 2001).
Interestingly, the three Brinker binding sites completely overlap the two Mad binding sites within the DRS. Indeed, the Dpp response critically depends on MadA; MadA fully overlaps Brk bs1, which is sufficient for Brinker function in vitro and in vivo. It was thus asked whether Brinker might be able to compete with Mad for DNA binding. Competitive DNA binding experiments were performed using bacterially expressed DNA binding domains of Brinker and Mad. This revealed that the former is capable of competing successfully with the latter for DNA binding at a molar ratio of 1:150, and Brinker almost completely blocks Mad binding at a ratio of 1:15. Note that full-length Mad binds to DNA less efficiently than its isolated DNA binding domain, indicating that Brinker would be able to compete even more successfully with full-length Mad. Thus, Brinker can block Mad binding to DNA in vitro in the presence of a considerable molar excess of Mad (Saller, 2001).
To confirm that the above Brinker binding sites within Ubx are functional targets in vivo, Brinker was expressed throughout the midgut mesoderm with the GAL4 system. This revealed that expression of Ubx in the middle midgut is nearly eliminated in Brinker-overexpressing embryos. Instead, many of these embryos show an endodermal bulge in the middle midgut that is also observed in Ubx mutants. Furthermore, the first and second midgut constrictions are rudimentary at best, and often missing altogether. Again, loss of the second constriction is indicative of mutations of Ubx and dpp, while loss of the first may reflect mutation of the dpp-related gene gbb. Finally, ectopic Brinker also drastically reduces dpp and wg expression in the middle midgut, which is expected since their expression depends on Ubx. This indicates that Brinker, by virtue of repressing Ubx, is capable of blocking the whole process of endoderm induction that depends on this HOX gene (Saller, 2001).
These results indicate that Brinker is a direct repressor of Ubx, and thus a potent antagonist of the Dpp-dependent process of endoderm induction. It is noted that Brinker is expressed in 'signal-free' zones bordering the anterior and posterior limits of the midgut. Its presence in these zones may have a barrier function, helping to block the spread of the Dpp response beyond the midgut limits (Saller, 2001).
Interestingly, the critical Brinker target site within Ubx B overlaps MadA, a functional Mad binding site that is required for the stimulation of this enhancer by Dpp signalling. Furthermore, Brinker competes effectively with Mad in binding to this site in vitro, and blocks activated Mad from stimulating Ubx B in vivo. This indicates that the mechanism by which Brinker repression dominates over stimulatory Dpp inputs is based on direct competition for binding to Dpp target enhancers. Given that most, if not all, Dpp signalling is mediated by Mad, it seems likely that this competition-based mechanism of Brinker repression is widespread and extends to genes that are Dpp targets in other developmental contexts (Saller, 2001).
Notably, MadA is also the target sequence for repression of Ubx B in response to high Wg levels in the middle midgut. MadA is thus a pivotal enhancer sequence that gauges and integrates positive inputs from Dpp and negative inputs from Brinker and Wg. Wg-mediated repression in the middle midgut is mediated by the zinc finger protein Teashirt and can be overriden by simultaneous Dpp stimulation. In contrast, Brinker-mediated repression dominates over simultaneous Dpp stimulation. It thus appears that Brinker is a more potent repressor than Teashirt, and is designed to function as a signal-antagonist even in the presence of high levels of Dpp signalling (Saller, 2001).
Brinker contains the sequence PMDLS, which resembles the P-DLS motif through which a number of transcription factors recruit the co-repressor dCtBP. Indeed, using in vitro pull-down assays, it was found that dCtBP binds to full-length Brinker as well as to an N-terminal Brinker fragment that contains the PMDLS motif. This suggests that Brinker may recruit dCtBP to repress Dpp target genes in the embryo. Interestingly, dCtBP assists various transcription factors, such as Knirps, Snail and Krüppel, that act at short-range to repress their target genes. These short-range repressors bind to autonomous enhancers to quench nearby bound transcriptional activators, which has prompted the suggestion that dCtBP may be specifically designed to quench. Therefore, this quenching ability of dCtBP could enable Brinker to not only compete efficiently with activated Mad in the binding of DNA, but also out-compete the activity of nearby transcription factors such as activated dTCF (Saller, 2001).
The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped (Sd), the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to
different signaling pathways. The obligate integration of selector and
signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory
networks during development (Guss, 2001).
Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).
These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).
Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]2 [Su(H)]2 element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]2 [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).
Morphogen gradients control body pattern by differentially regulating cellular behavior. Molecular events underlying the primary response to the Dpp/BMP morphogen have been analyzed in Drosophila. Throughout development, Dpp transduction causes the graded transcriptional downregulation of the brinker (brk) gene. Significance for the brk expression gradient is provided by showing that different Brk levels repress distinct combinations of wing genes expressed at different distances from Dpp-secreting cells. The brk regulatory region has been dissected and two separable elements have been identified with opposite properties, a constitutive enhancer and a Dpp morphogen-regulated silencer. Furthermore, genetic and biochemical evidence is presented that the brk silencer serves as a direct target for a protein complex consisting of the Smad homologs Mad/Medea and the zinc finger protein Schnurri. Together, these results provide the molecular framework for a mechanism by which the extracellular Dpp/BMP morphogen establishes a finely tuned, graded read-out of transcriptional repression (Müller, 2003).
The Dpp signaling system shapes an inverse profile of Brk expression, which serves as a mold for casting the spatial domains of Dpp target genes. Thus, the question of how the Dpp morphogen gradient is converted into transcriptional outputs can be largely reduced to the question of how Dpp generates an inverse transcriptional gradient of brk expression. An unbiased approach was applied to this problem by isolating the regulatory elements of brk. A protein complex has been identified and characterized that binds to and regulates the activity of these elements in a Dpp dose-dependent manner (Müller, 2003).
Dissection of the brk locus reveals two separable elements with opposite properties: a constitutive enhancer and a morphogen-regulated silencer. Both elements have a direct effect on the level of brk expression, and it is the net sum of their opposing forces that dictates the transcriptional activity of brk in any given cell. In this sense, expression of the brk gene behaves like a spring that is compressed by Dpp signaling. Its silencer and enhancer embody the variable compressing and constant restoring forces, respectively. As stated by Hooke's law, an increased elastic constant (e.g., two copies of the constitutive enhancer) either shifts the brk levels toward those normally present at more lateral positions or necessitates a correspondingly higher compressing force (e.g., more silencer elements or higher levels of Dpp signaling). Given the central role Brk plays in controlling growth and pattern together with the direct impact of the two regulatory elements on brk levels, it appears inevitable that their quantitative properties must exhibit a fine-tuned evolutionary relationship with each other and with those of the Dpp transduction system. It appears, furthermore, that both the brk enhancer as well as the brk silencer elements represent ideal substrates for evolutionary changes in morphology (Müller, 2003).
Based on combined genetic and biochemical analysis, it is proposed that upon Dpp signaling the following key players meet at the brk silencer elements to execute repression: the Smad proteins Mad and Med and the zinc finger protein Shn. The role of Shn must be to direct the signaling input provided by Mad and Med into transcriptional silencing. In principle, two scenarios can be envisaged by which Shn fulfills this task. Shn could possess repressor activity (presumably via recruitment of corepressors) but lack the ability to bind the brk silencer and, hence, depend on Mad/Med for being targeted to its site of action. Alternatively, Shn could be prebound to the silencer, but only be capable of recruiting corepressors upon interaction with Mad/Med. Based on the observation that a Shn/DNA complex cannot be detected in the absence of Mad/Med, the first of these two possibilities is favored. The molecular architecture of the protein complex binding to the brk silencer as well as the DNA sequences providing the specificity for the local setup of this complex remain to be determined in detail (Müller, 2003).
An additional protein, which appears to influence the events at the brk silencer, is Brk itself. Genetic experiments indicate that Brk negatively modulates its own expression, forming a short regulatory loop that contributes to the final shape of the Brk gradient. This autoregulatory action occurs also via the brk silencer element, suggesting that Brk directly participates in the protein-protein or protein-DNA interactions at this site (Müller, 2003).
Most regulatory events ascribed to Smad proteins to date concern signaling-induced activation of target gene transcription. In the case of the brk silencer Shn could be regarded as a “switch factor” that converts an inherently activating property of Smad proteins into transcriptional repression activity. Indeed, it has been shown that Smad proteins have the ability to recruit general coactivators with histone acetyl transferase activity. However, in an alternative and more general view, Smad proteins per se may provide no bias toward activation or repression. Their main function may be to assemble transcriptional regulatory complexes involving other DNA binding proteins and endow these complexes with additional DNA binding capacity. Such associated DNA binding factors would not only determine target site specificity, but, by their recruitment of either coactivator or corepressor proteins, also define the kind of regulatory influence exerted on nearby promoters. Since Shn directs Mad/Med activity toward repression, the existence of at least one other such Mad/Med partner in Drosophila is hypothesized to account for Mad/Med-mediated activation of gene expression. Such Mad/Med-mediated activation appears to be required for peak levels of sal and vg transcription, as well as for defining gene expression patterns in domains where brk expression is completely repressed, e.g., close to the Dpp source of the dorsal embryonic ectoderm (Müller, 2003).
At the heart of the model is the direct causal relationship between the formation of a Shn/Mad/Med/brk-silencer complex and the silencing of brk gene transcription. Although the two observations have been derived from different experimental data sets (biochemical versus genetic, respectively), there is a firm correlation between the requirements for either event to occur. brk is not repressed when either (1) the brk silencer elements are lacking or mutated; (2) or when Dpp input is prevented (and hence Mad is neither phosphorylated, nor nuclearly localized, nor associated with Med), or when (3) Shn is not present or is deprived of its C-terminal zinc fingers. The same set of requirements was observed for the formation of the Shn/Mad/Med/brk complex. Moreover, it is the concurrence of all three of these conditions that appears to provide the exquisite specificity to the Dpp-regulated silencing of gene transcription. (1) It only occurs in conjunction with a functional brk silencer, or an equivalent element. (2) There is an absolute requirement for Dpp input in Shn-mediated silencing. Not even a partial repressor activity of Shn was observed in cells that do not receive Dpp signal (e.g., loss of shn function in cells situated in lateral-most positions of the wing disc does not cause a further upregulation of brk transcription). (3) Shn represents only one of several zinc finger proteins expressed in Dpp receiving cells, yet none of the other proteins is able to substitute for Dpp-mediated repression. A major determinant for the specificity with which Shn engages in the signaling-dependent protein/DNA complex appears to be the triple zinc-finger motif. Although it is likely that this structural feature is required for contacting specific nucleotides on the brk silencer, the possibility cannot be not excluded that some of the zinc fingers mediate protein-protein interactions between Shn and Mad, Med or other cofactors (Müller, 2003).
Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. The Dpp/Tgfß signal controls Abdominal A function, and Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity (Grienenberger, 2003).
AbdA is expressed and is active in the third and fourth compartments of the midgut (PS8-PS12), and yet it activates the wg target gene only in PS8. Dpp secreted from PS7 is shown to provide the spatial information required for PS8-localized wg activation and, acting through a newly identified 546 bp enhancer, AbdA and Mad, a transcriptional effector of the Dpp pathway, directly control wg transcription. The convergence of Hox function and Dpp signaling therefore occurs at the levels of DNA and transcription, and endows AbdA with PS8-specific regulatory properties (Grienenberger, 2003).
To identify the enhancer responsible for wg expression in the VM,
subfragments of a 9kb genomic region known to drive wg embryonic
expression were
analyzed in transgenic lines transformed with lacZ reporter
constructs. The
smallest fragment that drives accurate expression in the VM is a 546 bp
XhoI/ClaI (XC) restriction fragment. Its activity is first
detected during germ-band retraction, when wg transcripts are visualized in the VM by in
situ hybridization,
and only in PS8 VM cells. During subsequent development, XC enhancer activity
still mimics wg expression, and is associated with the site of central midgut
constriction formation. Thus, from early on to the end of embryogenesis, the XC
enhancer exclusively and accurately recapitulates wg spatiotemporal
expression in the VM (Grienenberger, 2003).
To address whether AbdA and Dpp signaling could directly regulate wg, the sequence of the XC enhancer was examined for the presence of putative binding sites for AbdA and for Mad/Medea (referred to as DRS, for Dpp response sequence), the canonical transcriptional effectors of the Dpp signaling pathway known to recognize identical target sequences. Since genetic and molecular data led to the proposal that, in Drosophila, the CRE sequences to which Creb proteins bind are required to respond to Dpp in addition to DRSs, potential Creb binding sites were sought. Six TAAT core sequences and four sequences resembling the consensual Hox/Pbx binding sites (TGATNNATG/TG/A) were identified as potentially mediating AbdA function. The Hox/Pbx 3 and 2 sequences strongly match the consensus, with seven or six of the eight consensus nucleotides conserved, respectively. Hox/Pbx sequences 1 and 4 only have five of the eight consensus nucleotides conserved. The XC fragment contains three sequences matching DRSs and two potential CRE sites (Grienenberger, 2003).
To assess the evolutionary conservation of the XC enhancer, an homologous fragment from Drosophila virilis was isolated and analyzed for its in vivo activity by transgenesis in Drosophila melanogaster. The D. virilis fragment drives expression in a pattern very similar to that of the XC enhancer, suggesting that sequences conserved between these two enhancers may be important for wg regulation in the midgut. Sequence comparison, including sequences from D. pseudoobscura, revealed that a majority of the TAAT core motifs, the DRSs and the putative Creb-binding sequences are evolutionarily conserved, whereas sequences that match heterodimeric Hox/Pbx consensus binding sites are not. The existence of two large conserved sequences, Box 1 and 2, is noted. Since Box1 lies in a fragment that does not drive reporter gene expression in transgenic flies, particular attention was paid to Box2 (Grienenberger, 2003).
Hox signaling integration was examined to determine whether signaling pathways contribute towards specifying how AbdA, a widely expressed Hox selector protein, controls the development of distinct pattern elements at different locations. Dpp signal secreted from PS7 provides the positional cue responsible for localized activation of wg by AbdA. Biochemical and reverse genetics experiments have established that AbdA and Mad directly regulate wg transcription through the XC enhancer, which thus serves as an integrator of Hox and Dpp input. AbdA is impotent with respect to this enhancer in the absence of the Dpp signal, though it can function perfectly well on other genes without Dpp. Therefore, functional interactions between selector proteins and signaling pathways confer specificity to signaling pathways, and reciprocally confer functional diversity to selector proteins (Grienenberger, 2003).
This study provides a conceptual framework for understanding the molecular basis of regional Hox protein transcriptional activity. Dpp and Wg signaling subdivide the AbdA Hox domain, allowing activation of pointed (pnt) and opa target genes in the third and fourth midgut chambers, respectively. Based upon the data presented here, it is suspected that the localized activation of pnt and opa by AbdA also relies on direct enhancer integration of Hox and signaling inputs. Accordingly, a Hox/signaling combinatorial code functionally subdivides the domain where a single Hox protein is made, giving rise to discrete patterns of target gene activation. The structures of relevant cis-regulatory regions of AbdA target genes are instrumental for determining which signal is required to allow activation by AbdA. The pnt midgut enhancer would contain AbdA and Wg response elements and would be activated by AbdA specifically in the third midgut chamber through the combinatorial action of AbdA and the Drosophila Tcf/Arm transcriptional effector of Wg signaling. Similarly, the opa midgut enhancer would contain AbdA and Dpp response elements and would be activated only in the fourth gut chamber by AbdA, in this case because of an inhibitory effect of the Dpp-regulated transcription factor on AbdA activity (Grienenberger, 2003).
Further studies are required to understand how Hox selector proteins functionally interact with nuclear effectors of signaling pathways to generate specific transcriptional patterns. In the control of wg by AbdA, several scenarios can be envisioned. In one, the effect of the Dpp transcriptional effector Mad on AbdA activity would be indirect, by antagonizing the function of a repressor that would otherwise act on the XC enhancer to prevent wg expression. The absence of a binding site for this hypothetical repressor in Box2 could explain how Box2 drives AbdA-dependent transcription even without Dpp transcriptional effector binding sites. In a second scenario, Dpp transcriptional effectors would more directly control the activity of AbdA by influencing its DNA binding or transregulatory properties. A direct interaction of HoxC8 and Smad1 has been reported to induce osteoblast differentiation in mammals, suggesting that the coordinate action of AbdA and Dpp signaling might rely on direct AbdA-Mad interaction. In wg regulation, the situation may be different, as additional regulatory inputs are involved. bin and hth are essential, and Wg signaling is required for accurate levels of wg expression. The contribution of Creb might indicate that the Ras/Mapk signaling pathway is involved as well. Ras signaling has been proposed to play a permissive role by acting on CRE sequences of the Ubx and lab enhancers. These observations suggest that AbdA and Hox proteins in general attain specificity and diversity by participating in a variety of protein interactions in enhancer-binding complexes (Grienenberger, 2003).
The gene homothorax is required for the nuclear import of Extradenticle, The functions of exd/hth and of the
Hh/Wg/Dpp pathway are mutually antagonistic: exd blocks the response
of Hh/Wg/Dpp target genes such as optomotor-blind and
dachshund; high levels of Wg and Dpp eliminate exd function by repressing hth. This
repression is mediated by the activity of Dll and dac.
One prerequisite for
appendage development is the inactivation of the exd/hth
genes (Azpiazu, 2000 and references therein).
htx is originally expressed
uniformly in the wing imaginal disc but, during
development, its activity is restricted to the cells that form
the thorax and the hinge, where the wing blade attaches to
the thorax, and it is eliminated in the wing pouch, which forms
the wing blade. Repression of hth in the wing
pouch is a prerequisite for wing development; forcing hth
expression prevents growth of the wing blade. Both the Dpp
and the Wg pathways are involved in hth repression. Cells
unable to process the Dpp signal (lacking thick veins or Mothers against Dpp activity) or the Wg signal (lacking dishevelled
function) express hth in the wing pouch. vestigial has been identified as a Wg and Dpp response factor
that is involved in hth control. In contrast to its repressing
role in the wing pouch, wg upregulates hth expression in
the hinge; teashirt is
a positive regulator of hth in the hinge. tsh plays a role
specifying hinge structures, possibly in co-operation with
hth (Azpiazu, 2000).
In the second instar wing disc, the Hth product accumulates
uniformly in the thoracic and appendage regions of the disc, but throughout the third larval period hth expression
is downregulated and, by the late third instar, Hth only appears
in the presumptive regions of the thorax and the wing hinge.
The central part of the disc, which gives rise to the wing pouch,
shows no hth expression. The repression of hth
function is important for wing development, because if hth
activity is forced in the wing pouch, the wing does not form. A similar observation has been made in the leg disc;
hth or exd expression in the distal part results in a truncated
appendage in which all the distal components are missing. In the leg, the
subdivision between distal and proximal regions results from
the antagonism between Hh signaling and exd/hth function. Hh response genes such as Dll and dac are instrumental in repressing hth (Azpiazu, 2000 and references therein).
The downregulation of hth in the wing
pouch is a consequence of the activity of the Dpp and the Wg
signaling pathways. In cells in which the response to the Dpp
signal is prevented, as in tkv or Mad mutant cells, hth is
expressed at high levels. Similarly, dsh minus cells, in which
the transduction of Wg is blocked, show ectopic hth activity and
consequently nuclear exd expression. These results also indicate
that hth is latently active in the wing cells and has to be repressed
by the continuous activity of the Dpp and Wg signals. The
inability of cell clones to proliferate, cells in which the Dpp or the Wg pathways
have been totally eliminated, may be due to high
levels of hth expression. The Dpp
and Wg pathways repress hth expression independently. This is
illustrated by the experiments inducing dsh mutant clones:
ectopic hth expression is only observed in clones located away
from the AP border. This suggests that the high levels of
Dpp expression near the AP border are sufficient to impede hth
expression despite the removal of the control by Wg (Azpiazu, 2000).
Extracellular signals can act at different threshold levels to elicit distinct transcriptional and cellular responses. The transcriptional regulation of the Wingless target gene Ultrabithorax has been examined in the embryonic midgut of Drosophila. Ubx transcription is stimulated in this tissue by Dpp and by low levels of Wingless signaling. High levels of Wingless signaling can repress Ubx transcription. The response sequence within the Ubx midgut enhancer required for this repression coincides with a motif required for transcriptional stimulation by Dpp, namely a tandem array of binding sites for the Dpp-tranducing protein, Mad. Indeed, Wingless-mediated repression depends on low levels of Dpp, although apparently not on Mad itself. In contrast, high levels of Dpp signaling antagonize Wingless-mediated repression. This suggests that transcriptional activation of Ubx is subject to competition between Dpp-activated Mad and another Smad whose function as a transcriptional repressor depends on high Wg signaling. Wingless can repress its own expression via an autorepressive feedback loop that results in a change of the Wingless signaling profile during development (Yu, 1998).
Dpp and Wg signaling synergize in the visceral mesoderm to stimulate Ubx
transcription, targeting distinct, albeit adjacent, response sequences in the Ubx midgut enhancer. Therefore,
efficient stimulation of Ubx transcription by Wg depends on dpp.
Wg-mediated repression also depends on dpp, but, remarkably in this case, the response sequence for Wg-mediated repression within the Ubx enhancer coincides with that for Dpp-mediated stimulation.
Indeed, the WRS-R/DRS (Wingless response sequence mediating repression and Dpp response sequence) functions in two antipodal responses: it mediates efficient transcriptional
stimulation when the signaling levels of Dpp are high and those of Wg are low, but it is also required
for transcriptional repression when the Wg signaling levels are high and those of Dpp are low. This raises the possibility that the same factor may confer the two antipodal responses.
However, this is unlikely to be the case since Mad itself, which binds to the DRS to mediate the
positive response to Dpp, is apparently not required
for the Wg-mediated repression (Yu, 1998).
Thus it is proposed that the two antipodal responses are conferred by two distinct factors: by Mad and by
a hypothetical protein WR. It is further proposed that WR is a Mad-related protein, i.e. a
Smad, since WR acts through Mad-binding sites and since its function as a repressor depends on dpp.
It is envisaged that WR, like Mad itself and other Smads, is activated by Dpp signaling through
phosphorylation by ligand-bound membrane receptors, an event that promotes their subsequent
translocation to the nucleus. In this scenario, Dpp enables WR (which
also needs to be activated by high Wg signaling) to occupy the Mad-binding sites within the
Ubx enhancer. Once bound to this enhancer, WR dominantly represses Ubx transcription, overriding
the activating function of Arm-Pangolin and other transcriptional activators bound to the same enhancer (Yu, 1998).
How is WR's repressor function activated by high Wg levels? It is presumed that high Wg signaling
regulates, directly or indirectly, the availability of WR as an enhancer-binding protein: either high Wg
signaling controls a post-transcriptional event (e.g. it may promote WR's association with Armadillo, or
WR's translocation into the nucleus), or it simply activates transcription of the WR gene. The
latter possibility of indirect regulation, which involves transcriptional coupling, is favored because it accomodates
readily the dependence of Wg-mediated repression on arm and Pangolin. Whatever the case,
it is emphasized that high Wg signaling controls the activity of the protein WR (possibly a
Smad), which also requires Dpp signaling. Thus, WR is a common target for two signaling pathways
and represents a point of convergence between them (Yu, 1998).
This model readily explains how high Dpp levels antagonize WR, namely by promoting maximal levels
of nuclear Mad which now competes with WR for binding to the Ubx enhancer. The outcome of this
competition is the transcriptional activation or repression of target genes, depending on the prevalence
of Mad or WR. This may illustrate a general principle, namely that the response sequence
for the positive effect of one signal is also the response sequence for the negative effect of an
antagonistic signal. Such a layout provides a sharp flipping of the response from positive to negative in
an area where cells are experiencing increasingly more of one signal and increasingly less of the
antagonizing one (Yu, 1998).
Medea is the Smad4 homolog that is known to be the common oligomerization partner for pathway-specific Smads. Furthermore, Medea binds to the same DNA sequences as Mad. This raises the possibility that Medea is an oligomerization partner of WR: while Medea, together with Mad, is expected to activate transcription, together with WR it may repress transcription. A precedent for this scenario is the Myc/Mad/Max system, in which Mad (a bHLH protein that happens to have the same name as the Dpp transducer Mad) is a common dimerization partner for either Myc, a transcriptional activator, or Max, a transcriptional repressor. In addition to antagonism, there is also synergy between Wg and Dpp in the
embryonic midgut. This synergy apparently results from
cooperation between the nuclear target factors activated by the two signals, i.e. between Arm-Pangolin
and Mad/CRE-binding proteins. Other examples of apparent synergy between Wg and Dpp are the leg and wing imaginal discs,
where these signals act together in central disc regions to stimulate expression of homeobox genes. But the two signals also antagonize
each other in leg discs, as well as in eye discs. Although it is conceivable that the developmental context
determines the synergy or antagonism between Dpp and Wg, the situation in the midgut suggests that
the decisive factor in each case may be the levels of signaling (Yu, 1998 and references).
It is interesting that Wg signaling can repress its own expression when signaling levels reach a
critically high level. This indicates a negative feedback loop, which could account for two observations: (1) Wg signaling shifts its own expression towards the anterior over time. It is not known at present whether this shift has any biological significance. (2) Wg has the potential for
switching itself off over time. This is actually observed, since Wg expression becomes undetectable by the
end of embryogenesis. Clearly, Wg's negative feedback loop is capable of changing the Wg
signaling profile as development procedes. There are negative feedback loops for other signaling pathways in Drosophila. For example, the
epidermal growth factor (EGF) receptor inhibits itself eventually, after signaling has reached a critical
level, by switching on expression of an inhibitory ligand, Argos. In the ovary, this negative feedback loop causes splitting of a single signaling peak into
twin peaks. Furthermore, Hedgehog signaling in the eye
imaginal disc is repressive at high Hedgehog levels, but stimulatory in cells, further away from the
signaling source, which experience lower Hedgehog levels. Perhaps such
'hard-wired' negative feedback loops in signaling pathways are fairly universal, and serve to stop these
pathways from escalating out of control. If so, this would be akin to feedback inhibition of metabolic
pathways, which provides homeostatic control (Yu, 1998 and references).
The Drosophila Vestigial protein has been shown to play an
essential role in the regulation of cell proliferation and
differentiation within the developing wing imaginal disc.
Cell-specific expression of vg is controlled by two separate
transcriptional enhancers. The boundary enhancer
controls expression in cells near the dorsoventral (DV)
boundary and is regulated by the Notch signal transduction
pathway, while the quadrant enhancer responds to the
Decapentaplegic and Wingless morphogen gradients
emanating from cells near the anteroposterior (AP) and DV
boundaries, respectively. MAD-dependent activation of the
vestigial quadrant enhancer results in broad expression
throughout the wing pouch but is excluded from cells near
the DV boundary. This has previously been thought to be
due to direct repression by a signal from the DV boundary;
however, this exclusion of quadrant enhancer-dependent
expression from the DV boundary has been shown to be due to the
absence of an additional essential activator in those cells.
The Drosophila POU domain transcriptional regulator,
Drifter, is expressed in all cells within the wing pouch
expressing a vgQ-lacZ transgene and is also excluded from
the DV boundary. Viable drifter hypomorphic mutations
cause defects in cell proliferation and wing vein patterning
correlated with decreased quadrant enhancer-dependent
expression. Drifter misexpression at the DV boundary
using the GAL4/UAS system causes ectopic outgrowths at
the distal wing tip due to induction of aberrant Vestigial
expression, while a dominant-negative Drifter isoform
represses expression of vgQ-lacZ and causes severe
notching of the adult wing. In addition, an essential evolutionarily conserved sequence element
bound by the Drifter protein with high affinity has been identified and it has been located adjacent to the MAD binding site within the quadrant
enhancer. These results demonstrate that Drifter functions
along with MAD as a direct activator of Vestigial expression
in the wing pouch (Certel, 2000).
Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and
Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting
both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects
of three signal-activated transcription factors (Pangolin/dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and
Tinman) on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman
determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).
Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial
signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess
information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and
founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).
Previous genetic experiments have defined multiple intercellular signaling events that govern the progressive determination of the Eve progenitors. Signaling from both the Wnt family member Wingless (Wg) and the TGF family member Decapentaplegic (Dpp) prepatterns the
mesoderm and renders cells competent to respond to Ras/MAPK activation. Localized Ras activation within the competence domain determined by the
intersection of Wg and Dpp expression occurs through the action of two RTKs: the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl)
fibroblast growth factor receptor. This RTK signaling induces two distinct equivalence groups, each of which expresses Eve. Lateral inhibition mediated by
Notch then selects a single progenitor from each equivalence group (Halfon, 2000).
The present study explores how the prepattern genes wg and dpp establish competence for mesodermal cells both to activate and to respond to the
Ras/MAPK cascade; how multiple intercellular signals are integrated to establish Eve progenitor fates, and how muscle- and cardiac-specific responses to Ras
signaling are generated. Wg provides competence for the generation of the Ras/MAPK inductive signal by regulating the expression of key
proximal components of the Egfr and Htl RTK pathways. Wg and Dpp then create competence for a specific response to the inductive signal both through their
own respective downstream transcriptional effectors, dTCF and Mothers against
dpp (Mad), and through their regulation of the mesoderm-specific transcription factors Tinman (Tin) and Twist (Twi). Specificity of the Ras/MAPK response is achieved though the
integration of these signal-activated and tissue-restricted transcription factors, along with the Ras/MAPK-activated Ets domain transcription factor PointedP2
(Pnt), at a single transcriptional enhancer. These results provide a direct link between the initial axis patterning processes in the early embryo and the subsequent
combinatorial signaling events that lead to the progressive determination of muscle and cardiac progenitors (Halfon, 2000).
The Eve progenitors in each mesodermal hemisegment arise during embryonic stage 11 in a dorsal region demarcated by the intersecting domains of Wg and Dpp expression. The cells exposed to both Wg and Dpp are competent to respond to localized Ras signaling, which induces the initial expression of Eve in two clusters of equipotent cells. In each of these equivalence groups, activity of the Notch pathway leads to the rapid refinement of Eve expression to a single muscle or cardiac progenitor. The two Eve equivalence groups arise sequentially. Cluster C2, from which progenitor P2 derives, is first to form. P2 divides asymmetrically, with one daughter maintaining Eve expression and becoming the founder of the two EPCs (F2EPC), and the other losing Eve expression and becoming the founder of muscle DO2. The second Eve-expressing cluster, C15, forms slightly later and produces the progenitor P15, which in turn divides to yield the founder of the Eve-expressing muscle, DA1, and an Eve-negative cell of as-yet-undetermined identity. Activation of the Ras/MAPK pathway in C15 depends on both the Egfr and Htl RTKs, but only Htl signaling is required for C2 formation (Halfon, 2000).
Next to be determined was at what level in the RTK/Ras pathway Wg is required for MAPK activation. In wg mutant embryos, there is loss of (1) the P2-specific expression of Htl; (2) its specific downstream signaling component, Heartbroken (Hbr, also known as Dof and Stumps), and (3) Rhomboid (Rho), a protein involved in the presentation of the Egfr ligand Spitz. Conversely, constitutive Wg signaling, achieved by ectopic expression of Wg or an activated form of the downstream Wg pathway component Armadillo (Arm), induces Htl, Hbr, and Rho expression in more dorsal mesodermal cells than the single P2 progenitor found at a comparable developmental stage. This effect is less prominent for Rho than for Htl and Hbr, which may reflect different threshold responses to Wg. Alternatively, the effect on Htl and Hbr may be more pronounced because ectopic Wg signaling prolongs their earlier expression in the entire C2 cluster; Rho, in contrast, is normally expressed in P2 but not in C2, possibly making it more refractory to a prepattern factor such as Wg. Expanded expression of these RTK pathway components is associated with increased MAPK activation and Eve expression. However, these effects of Wg hyperactivation are transient, with a normal number of Eve progenitors eventually segregating. Moreover, activated Arm is able to fully rescue Htl, Hbr, Rho, diphospho-MAPK, and Eve expression in wg mutant embryos. Htl, Hbr, and Rho expression, as well as MAPK activation, are also Dpp dependent. In summary, Wg and Dpp regulate the production of several key proximal components of the Egfr and Htl signal transduction pathways (Halfon, 2000).
One mechanism that would ensure the convergence of the multiple regulatory inputs required for the formation of P2 and P15 is integration by a transcriptional enhancer. Since Eve expression is the feature that uniquely identifies these progenitors, an investigation was made of whether eve itself is a direct target for regulation by both signal-activated and tissue-specific transcription factors. Regulatory sequences responsible for mesodermal eve expression are located approximately 6 kb downstream of the transcription start site. Deletions of this region were generated and a 312 bp minimal enhancer was defined that has been termed the eve Muscle and Heart Enhancer (MHE). When fused to a nuclear-lacZ reporter gene, the MHE drives expression in a mesodermal pattern identical to that of the endogenous eve gene. Reporter expression initiates at early stage 11, coincident with the onset of Eve expression in the equivalence group C2. Following formation of P2, MHE activity is observed in P15 and in the P2 daughters, F2EPC and F2DO2, then in the EPCs and the F15 daughters of P15, and finally in muscle fiber DA1. Colocalization of MHE-driven ß-galactosidase expression with Runt, which marks the F2DO2 founder and muscle DO2, establishes that the reporter gene expression present in Eve-negative sibling cells is a result of ß-galactosidase perdurance. Of note, the MHE mimics endogenous Eve expression despite its lack of a consensus binding site for the transcription factor Zfh-1 that had previously been proposed to play a role in mesodermal eve regulation (Halfon, 2000).
Strikingly, the MHE is only active in a single nucleus of the mature DA1 and DO2 muscles. It is inferred that these are the original nuclei of the F15DA1 and F2DO2 founders based on prior reporter expression in those cells. Similar results were obtained when DNA flanking the MHE by several hundred base pairs on either side (+4.96 to +7.36 kb), including the previously described Zfh-1 site, was included in the reporter construct, or when the MHE was placed 3' to a reporter gene fused to the endogenous eve promoter. Thus, additional sequences are required for eve expression in non-founder myofiber nuclei. Of critical importance to the present study, the MHE fully recapitulates mesodermal Eve expression during the signal-dependent induction of progenitor and founder cells (Halfon, 2000).
Genetic manipulation of the Wg, Dpp, and RTK/Ras signaling pathways causes predictable alterations of endogenous mesodermal Eve expression. A determination was made of whether the isolated MHE responds appropriately to these signals. In all genetic backgrounds, reporter gene expression corresponds precisely to that of endogenous eve. For example, constitutively activated Arm transiently increases the expression of both genes. However, Wg hyperactivation does not have a stable effect on MHE function. In contrast, both endogenous eve and the MHE-driven reporter are induced throughout the initial competence domain by constitutively activated Pnt, and expression of both markers extends laterally in the presence of activated Arm plus Pnt. Ectopic Dpp leads to both endogenous Eve and MHE-driven reporter expression in the ventral mesoderm, while coexpression of Dpp and activated Ras1 induces expression of both genes in a dorsal-ventral stripe. These results demonstrate that the isolated MHE is responsive to all of the known signals that are essential for the specification of Eve progenitors (Halfon, 2000).
Given that the MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).
To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).
It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).
Stem cells execute self-renewing and asymmetric cell divisions in close association with stromal cells that form a niche. The mechanisms that link stromal cell signaling to self-renewal and asymmetry are only beginning to be identified, but Drosophila oogenic germline stem cells (GSCs) have emerged as an important model for studying stem cell niches. Decapentaplegic sustains ovarian GSCs by suppressing differentiation in the stem cell niche. Dpp overexpression expands the niche, blocks germ cell differentiation, and causes GSC hyperplasty. The bag-of-marbles (bam) differentiation factor is the principal target of Dpp signaling in GSCs; ectopic bam expression restores differentiation even when Dpp is overexpressed. The transcriptional silencer element in the bam gene integrates Dpp control of bam expression. Finally and most significantly, this study demonstrates that Dpp signaling regulates bam expression directly since the bam silencer element (SE) is a strong binding site for the Drosophila Smads, Mad and Medea. These studies provide a simple mechanistic explanation for how stromal cell signals regulate both the self-renewal and asymmetric fates of the products of stem cell division (Chen, 2003).
GSCs divide in the anterior/posterior axis, and this division produces daughters with different fates. The anterior cell of a GSC division retains contact with the stromal cap cells, maintains high levels of Dpp signaling, and continues as a stem cell. The posterior stem cell daughter dissociates from the cap cells and becomes a cystoblast (CB). The CB divides precisely four times with incomplete cytokinesis, giving rise to a cyst of 16 interconnected cells that differentiate further into 1 oocyte and 15 nurse cells. The progress of cyst formation can be followed by monitoring the morphogenesis of a dynamic organelle, termed the fusome, that grows and branches with each cyst cell mitosis (Chen, 2003).
Cystoblasts require the product of the bag-of-marbles (bam) gene, which is both necessary and sufficient for differentiation. Germ cells lacking bam fail to differentiate into cystoblasts and continue to divide with full cytokinesis, producing germ cell hyperplasia. The failure of these proliferating germ cells to differentiate can be recognized by following the fusome since it remains spherical instead of growing into a branched structure. Superficially, therefore, bam mutant cells behave like GSCs, but molecular markers to determine the stage of arrest have been lacking (Chen, 2003).
Studies characterizing bam transcription demonstrate that bam is tightly regulated such that it is off in GSCs and on in CBs. Thus, it was possible to determine if bam mutant cells are 'stem cell-like' since the activity of a bam reporter transgene would distinguish GSCs from CBs precisely. GFP expression was examined in bam mutant animals carrying a transgene with the bam promoter fused to GFP; most germ cells were observed to be GFP positive. Thus, unlike GSCs, germ cells lacking bam advanced to a state of differentiation sufficient to activate bam transcription (Chen, 2003).
Two features of GSC divisions demand molecular explanation: how is the anterior daughter of the GSC division retained as a stem cell (self-renewal) and what causes the posterior daughter to differentiate into a CB (asymmetric division)? Stromal cells, including the cap and inner sheath cells, at the germarial tip express several signaling molecules and are a likely source of dpp. dpp signaling has been shown to be required to maintain GSCs, and transcriptional control over Bam has been shown to distinguish GSCs from CBs. These two phenomena can now be linked directly (Chen, 2003).
In GSCs, in which dpp signaling and pMad levels are highest, the Mad:Med complex binds to the bam SE and prevents bam transcription. GSCs self-renew because association of the anterior daughter with stromal cells permits sufficient dpp signaling to block CB differentiation by assembling a repressor complex on the bam SE element. This complex is likely to include other factors required for transcriptional antagonism, such as TGIF, a homeodomain-containing transcriptional corepressor of TGF-beta-dependent gene expression, or Ski/Sno factors, which can recruit histone-modifying enzymes. The complex may also contain Schnurri, a negatively acting Mad cofactor, since shn mutant GSCs also differentiate precociously (Chen, 2003).
During division, a GSC daughter cell is displaced away from the cap cells and into a region of diminished dpp signaling. This cell, the CB precursor (pre-CB), expresses lower pMad levels that would cause the concentration of Mad:Med complexes to fall. Declining occupancy levels of the bam SE would produce derepression of bam transcription and concomittant activation of the CB differentiation program (Chen, 2003).
Embryonic stem cells are considered totipotent because they can populate any of the adult niches. Although the degree of adult stem cell plasticity is currently receiving much attention, assembly of stem cells into signaling niches during postembryonic development might impose differentiation limits. What are the specific effects of stromal cell niches on captured stem cells? In the case of the Drosophila ovarian niche, GSCs are maintained as 'CBs-in-waiting' because a stromal cell signal represses the expression of one key factor (i.e., Bam). Perhaps other types of stem cells are similarly differentiated but blocked by stromal cell signals and require the expression of only one or a few key molecules to resume development (Chen, 2003).
The Drosophila ovary is an attractive system to study how niches
control stem cell self-renewal and differentiation. The niche for germline
stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC
maintenance. bam is both necessary and sufficient for the
differentiation of immediate GSC daughters (cystoblasts). Bmp
signals directly repress bam transcription in GSCs in the
Drosophila ovary. Similar to dpp, gbb encodes another Bmp
niche signal that is essential for maintaining GSCs. The expression of
phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for two essential Bmp signal
transducers (Med and punt) bam transcription is also elevated. Finally, Med and Mad are shown to directly bind to the bam silencer in vitro. This
study demonstrates that Bmp signals maintain the undifferentiated or
self-renewal state of GSCs, and directly repress bam expression in
GSCs by functioning as short-range signals. Thus, niche signals directly
repress differentiation-promoting genes in stem cells in order to maintain
stem cell self-renewal (Song, 2004).
This study reveals a new function for gbb in the regulation of GSCs in the Drosophila ovary. Loss of gbb function leads
to GSC differentiation and stem cell loss, similar to dpp mutants.
gbb is expressed in somatic cells but not in germ cells, suggesting
that gbb is another niche signal that controls GSC maintenance. Like dpp, gbb contributes to the production of pMad in GSCs and also functions to repress bam expression in GSCs. As in the wing imaginal disc, gbb also probably functions to augment the dpp signal in the regulation of GSCs through common receptors in the Drosophila ovary. In both dpp and gbb mutants, pMad accumulation in GSCs is severely reduced but not completely diminished. Since the dpp or gbb mutants used in this study do not carry complete loss-of-function mutations, it remains possible that complete elimination of either dpp or gbb function is sufficient for eradicating pMad accumulation in GSCs. Alternatively, both dpp and gbb signaling are required independently for full pMad accumulation in GSCs, and thus disrupting either one of them only partially diminishes pMad accumulation in GSCs. The lethality of null dpp and gbb mutants, and the
difficulty in completely removing their function in the adult ovary, prevent these possibilities from being tested directly (Song, 2004).
Interestingly, dpp overexpression results in complete suppression of cystoblast differentiation and complete repression of bam transcription in the germ cells, whereas gbb overexpression does not have obvious effects on cystoblast differentiation or bam transcription. Even though the UAS-gbb transgene and the c587 driver for gbb overexpression have been demonstrated to function properly, it is possible that active Gbb proteins are not produced in inner sheath cells and somatic follicle cells because of a lack of proper factors that are required for Gbb translation and processing in those cells, which could explain why the assumed gbb overexpression does not have any effect on cystoblast differentiation. However, since active Dpp proteins can be successfully achieved using the same expression method, and Dpp and Gbb are closely related Bmps, it is unlikely that active Gbb proteins are not produced in inner sheath cells and follicle cells. Alternatively, dpp and
gbb signals could have distinct signaling properties, and
dpp may play a greater role in regulating GSCs and cystoblasts.
Recent studies have indicated that Dpp and Gbb have context-dependent
relationships in wing development. In the wing disc, duplications of dpp are able to rescue many but not all of the phenotypes associated with gbb mutants, suggesting that dpp and gbb have not only partly redundant functions but also distinct signaling properties. In the wing and ovary, gbb and dpp function through two Bmp type I receptors, sax and tkv. The puzzling difference between gbb and dpp could be explained by context-dependent modifications of Bmp proteins, which render different signaling properties in different cell types. It will be of
great future interest to better understand what causes Bmps to have distinct signaling properties (Song, 2004).
All the defined niches share a commonality, structural asymmetry, which
ensures stem cells and their differentiated daughters receive different levels of niche signals. In order for a niche signal to function differently in a stem cell and its immediately differentiating daughter cell that is just one cell away, it has to be short-ranged and localized. This study shows that Bmp signaling mediated by Dpp and Gbb results in preferential expression of pMad and Dad in GSCs. Bmp signaling appears to elicit different levels of responses in GSCs and cystoblasts, suggesting that the cap cells are likely to be a source
for active short-ranged Bmp signals. These observations support the idea that Bmp signals are active only around cap cells. Consistently, when GSCs lose contact with the cap cells following the removal of adherens junctions they move away from the niche and then are lost. As
gbb and dpp mRNAs are broadly expressed in the other somatic
cells of the germarium besides cap cells, localized active Bmp proteins around cap cells could be generated by localized translation and/or activation of Bmp proteins. As they can function as long-range signals, it
remains unclear how Dpp and Gbb act as short-range signals in the GSC
niche (Song, 2004).
Bmp signaling and
bam expression are in direct opposition in Drosophila ovarian
GSCs. bam is actively repressed in GSCs through a defined transcriptional silencer. These observations lead to a model in which Bmp
signals from the niche maintain adjacent germ cells as GSCs by actively
suppressing bam transcription and thus preventing differentiation
into cystoblasts. The levels of pMad are correlated with the
amount of bam transcriptional repression in GSCs and cystoblasts. In the wild-type germarium, pMad is highly expressed in GSCs and some cystoblasts where bam is repressed. In other cystoblasts and differentiated germline cysts, pMad is reduced to very low levels, and thus bam transcriptional repression is relieved. In the GSCs mutant for dpp, gbb or punt, pMad levels are severely reduced, and bam begins to be expressed. The repression of bam transcription as a result of dpp overexpression seems to be a rapid process; bam mRNA is reduced to below detectable levels two hours after dpp is overexpressed. This suggests that repression of bam transcription by Bmp signaling could be direct. Furthermore, Med and Mad can
bind to the defined bam silencer in vitro, which also supports the
idea that Bmp signaling acts directly to repress bam transcription.
Dpp signaling has also been shown to repress brinker (brk) expression in the wing imaginal disc and in the embryo. The
repression of brk expression by Dpp signaling is mediated by the
direct binding of Mad and Med to a silencer element in the brk
promoter. Since the brk silencer is very similar to the
bam silencer, the results suggest that bam repression in
GSCs is also mediated directly by Dpp and Gbb in a similar manner (Song, 2004).
It remains unclear how the binding of Med and Mad to the bam
silencer results in bam transcriptional repression in GSCs. For the
brk silencer, Dpp signaling and Shn are both required to repress
brk expression in the Drosophila wing disc and embryo. Mad and Med belong to the Smad protein family, which are known to function as
transcriptional activators by recruiting co-activators with histone
acetyltransferase activity. In the wing disc, Shn is proposed to function as a switch factor that converts
the activating property of Mad and Med proteins into a transcriptional
repressor property. Possibly, the Mad-Med complex could also recruit Shn to the bam repressor element. Consistent with the possible role of Shn in repressing bam expression in GSCs is the observation that GSCs that lose shn function differentiate, and thus are lost.
Also, it remains possible that Mad and Med could recruit a repressor other
than Shn when binding to the bam repressor element. In the future, it will be very important to determine whether Shn itself is a co-repressor for Mad/Med proteins or whether it directly recruits a co-repressor to repress bam transcription in GSCs (Song, 2004).
In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. Following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a 'PGC niche' (Gilboa, 2004).
The embryonic gonad in Drosophila forms when somatic gonadal cells encapsulate about 12 primordial germ cells on each side of the embryo. PGCs proliferate in the female gonad during the subsequent three larval stages until, at the late-third larval stage, the gonad carries over 100 germ cells. At the larval/pupal transition, PGCs at the posterior of the gonad differentiate. Similar to differentiating germ line stem cells, differentiating PGCs undergo several rounds of incomplete mitotic divisions to form cysts and subsequently egg chambers with one oocyte and 15 nurse cells. Differentiation of posterior PGCs at the larval/pupal transition was attributed to the hormonal changes that control pupa formation. At the anterior part of the gonad, the newly formed somatic niche prevents the differentiation of PGCs and these are maintained as self-renewing GSCs throughout adult life. It has therefore been proposed that the transition from PGCs to GSCs coincides with the larval/pupal transition and the formation of the somatic niche. This study examines the genetic mechanisms that control PGCs during the proliferative larval stages, to better understand the PGC to GSC transition (Gilboa, 2004).
To explore how PGC differentiation is inhibited in the larval ovary, tests were performed to see whether any of the genes that are needed for either GSC maintenance or differentiation in the adult are likewise required in PGCs. Bag of marbles (Bam) has been shown to be a critical differentiation factor, because it is both necessary and sufficient to induce adult GSCs to differentiate. Therefore whether overexpression of Bam in the larval ovary is sufficient to drive PGCs to differentiate and form cysts was tested. As a marker for cyst formation, an antibody against an adducin-like molecule (1B1) was used, that stains the fusome, a sub-cellular organelle that is spherical in PGCs and GSCs but extends and branches as the single germ cell forms a multicellular cyst. Larvae carrying the bam gene under control of a heat-shock promoter (hs-bam) were heat-shocked at various time points during larval development and their gonads were examined one or two days later. Control wild-type gonads at the larval/pupal transition (LL3) exhibited either single PGCs carrying a spherical fusome or 2-cell cysts, which shared a bar-like fusome. The latter could be PGCs undergoing division, prior to full dissociation, or PGCs that initiated differentiation at the larval/pupal transition. In contrast, in hs-bam gonads, PGCs differentiate, as shown by the many cysts with branched fusome. Differentiation in hs-bam ovaries depended on the time of heat-shock. No cysts were observed in ovaries derived from larvae that were heat-shocked at the end of embryogenesis. Heat-shocking at the end of the first-larval instar (LL1) led to a high fraction (75%) of gonads that carried many cysts, whereas heat-shocking at the end of the second-larval instar (LL2) led to differentiation of all PGCs in all gonads tested. Thus, PGCs are able to differentiate prior to the larval/pupal transition. The time-dependent response to hs-bam could indicate either that PGCs are more capable of differentiation as the animal matures or that transcription from the hs promoter may be more active in the later larval stages. In support of the latter hypothesis, PGCs are transcriptionally quiescent during early embryogenesis and acquire transcriptional competence as they start to migrate. Indeed, the quantity of bam transcript seems limiting because a less rigorous heat-shock regime induces fewer cysts. Furthermore, with a different expression system, PGCs could be induced to differentiate as early as the end of embryogenesis. In support of the notion that differentiating PGCs follow the normal differentiation program, it was found that the time course of mitotic divisions in cysts that were precociously induced at LL2 was similar to that observed in cysts during normal development in either the pupal or the adult ovary (Gilboa, 2004).
These results suggest that all PGCs in the larval ovary are capable of differentiating following overexpression of Bam. Therefore whether active repression is required to keep PGCs in a proliferative state was tested. In adult GSCs, the Decapentaplegic (Dpp) pathway plays a major role in GSC maintenance. Dpp is produced by niche cells and is perceived directly by GSCs. Dpp signaling activates the downstream components Mothers against dpp (Mad) and Medea (Med), which directly bind to the bam promoter and repress the transcription of bam. In the larval gonad, overexpression of Dpp induces overproliferation of PGCs, suggesting that PGCs can respond to a Dpp signal. However, PGCs have not been shown to require Dpp in larval gonads. It was found that abolishing Dpp signaling in PGCs by overexpression of the negative regulator Daughters against Dpp (Dad) or mutations in thickveins (tkv), the Dpp type I receptor, induced differentiation of PGCs. 16-cell cysts were observed already at LL2 (48 hr after hatching), suggesting that PGCs begin differentiation shortly after the end of embryogenesis. Oocyte determination was detected in LL3 gonads, as indicated by accumulation of Orb, an oocyte marker, in one cell of the cyst (Gilboa, 2004).
To further explore the requirement for Dpp within GSCs, it was asked whether Dpp signaling could be detected directly in larval PGCs by monitoring the accumulation of its target, phosphorylated Mad (pMad) in PGCs. In larval gonads all PGCs accumulate pMad in the nucleus, suggesting that during larval development all PGCs receive a Dpp signal that actively represses their differentiation. In the adult, only germ line cells close to the niche contain significant levels of nuclear pMAD. Thus, the larval ovary may function in a similar manner to the adult niche in the prevention of PGCs from differentiation (Gilboa, 2004).
GSC differentiation is repressed by extrinsic factors, such as Dpp, and also by intrinsic factors. To further test whether PGCs employ the same mechanisms as GSCs to repress differentiation, larval ovaries were examined that were mutant for the translational repressors Nanos (Nos) and Pumilio (Pum), which function within GSCs to repress their differentiation. Indeed, nos mutant LL3 gonads contained many developed cysts. pumilio (pum) mutant gonads also contained cysts, although less so than nos mutants. Gonads that were mutant for both nos and pum did not contain more cysts than gonads that were mutant for nos alone. Because the alleles that were used were very strong, this suggests that nos and pum function together in the repression of PGC differentiation (Gilboa, 2004).
In adult ovaries, the differentiation of cysts requires Bam, and increasing amounts of Bam are present during each subsequent mitotic division. A reporter construct of GFP under control of the bam promoter was used to follow bam expression in the larval cysts. Cysts found in nos ML3 larval gonads also expressed higher amounts of GFP as compared to single PGCs. As in adults, the intensity of GFP labeling corresponds to the developmental state of the cyst. In addition to precocious differentiation, nos mutant germ cells displayed aberrations in the shape of the branched fusome and increased amount of small fusomal material as compared with wild-type. It is concluded that both Nos and Pum, which are required for GSC maintenance, are also required to repress PGC differentiation (Gilboa, 2004).
To further test for a possible partnership between nos and pum in GSC maintenance, the time at which nos or pum mutant germ line clones, generated by the FLP-FRT method, were eliminated from the adult ovary was examined. In wild-type, clones of unmarked GSCs were induced in about 25% of the ovarioles and that percent decreased only slightly during the course of the experiment, probably due to the natural rate of GSC loss. nos and pum mutant GSCs, in contrast, were lost rapidly. GSC loss was observed as early as 4 days after clone induction, and by the 6th or 7th day, most ovarioles did not contain a mutant GSC. The striking similarity in the profiles of nos and pum GSC loss therefore suggests that these genes also function together within GSCs (Gilboa, 2004).
As of the fifth and sixth day after clone induction, it was found that many nos mutant cysts were eliminated from the ovary. These results agree with the death of cysts observed in nos and pum mutants and with the death of nos cysts in pupal ovaries, which may be the cause of the empty ovarioles observed in adult nos females. These results and the previously reported phenotypes of nos and pum suggest that these genes are continually required throughout germ cell life. In the embryo, nos and pum are required for correct migration, transcription, and viability. During larval stages, they are required for the repression of PGC differentiation and, in the adult, for the maintenance and viability of GSCs as well as for the viability of differentiating cysts (Gilboa, 2004).
The targets of Nos and Pum within GSCs remain elusive, and the relationship of these 'intrinsic' GSC maintenance factors to the 'extrinsic' Dpp signal is unclear. To test if Dpp could function partly through Nos, the Nos expression pattern was examined in wild-type and in tkv-mutant GSCs. In wild-type germaria Nos is expressed at intermediate levels in GSCs and their immediate daughters, at very low levels during mitotic divisions of the cyst, and at very high levels in a fraction of the 16-cell cysts. This expression pattern was unchanged in tkv-mutant germ cells. Similar results were obtained for larval PGCs; Nos was expressed at intermediate levels in wild-type and tkv mutant PGCs, at lower levels in cysts undergoing mitosis, and at very high levels in 16-cell cysts. This suggests that Nos expression is independent of Dpp signaling (Gilboa, 2004).
Next, whether nos is required for Dpp function was tested, by analyzing nos mutant PGCs that were overexpressing either Dpp or TkvQD, a constitutively activated form of Tkv. In nos mutant control gonads, fragmented fusomal material as well as branched cysts could be observed. The spherical fusome within nos mutant germ cells remained small or fragmented in nos gonads overexpressing Dpp. Most strikingly, single PGC/GSC like germ cells accumulated in these gonads, and no cysts could be found. Thus, although increased Dpp signaling cannot fully counteract the nos phenotype, it does prevent precocious differentiation of nos mutant PGCs. Similar results were obtained with PGCs expressing TkvQD. In most gonads no cysts could be observed, although occasionally a small branched fusome could be detected, suggesting that Dpp signaling acts directly on PGCs, rather than via a secondary signal. The genetic data show that PGCs that are mutant for nos, can still respond to a Dpp signal, which keeps them in an undifferentiated state (Gilboa, 2004).
During larval stages, PGCs proliferate rather than differentiate. The translational repressors Nos and Pum are required to repress PGCs differentiation during larval stages. It has also been show that the Dpp pathway functions in a similar manner. Both pathways are also required for GSC maintenance. The fact that the spherical fusome remains abnormal in nos mutant gonads even when Dpp is overexpressed may suggest that some of Nos function is downstream of Dpp. However, the Nos expression data and the fact that Dpp signaling can prevent nos mutant PGCs from differentiation are more compatible with the Nos pathway playing a role upstream or in parallel to the Dpp pathway. It remains unclear how these pathways converge within germ cells (Gilboa, 2004).
Germ cells may perceive a Dpp signal from the moment they form at the posterior pole of the embryo until they differentiate to form cysts. Indeed, pMad is present in embryonic pole cells, larval PGCs and adult GSCs. Dpp signaling is not only necessary for GSC maintenance but also required continually through larval stages to actively repress PGC differentiation. Thus, the larval ovary functions in a similar manner to the adult niche with regard to Dpp-mediated repression of differentiation. During the third-larval instar, the adult somatic niche forms, and repression of PGC differentiation may then become limited to the small area of the adult ovary, allowing PGCs outside the confinement of the niche to differentiate (Gilboa, 2004).
Repression of PGC differentiation is required for about 4 days, from the end of embryogenesis to the beginning of pupa formation, whereas GSCs are maintained in the adult for many days. Differences between the 'short-term' and the 'long-term' repression of differentiation may yet be found. However, all the genes tested, dpp, bam, nos, and pum, function similarly in GSCs and PGCs. This similarity suggests that there may not be a clear transition from a 'dividing' PGC to a 'self-renewing' GSC (Gilboa, 2004).
The sensory organs of the Drosophila adult leg provide a simple
model system with which to investigate pattern-forming mechanisms. In the leg, a group of small mechanosensory bristles is organized into a series of
longitudinal rows, a pattern that depends on periodic expression of the
hairy gene and the proneural genes achaete
and scute. Expression of ac in
longitudinal stripes in prepupal leg discs defines the positions of the
mechanosensory bristle rows. The ac/sc expression domains
are delimited by the Hairy repressor, which is itself periodically expressed. In order to gain insight into the molecular mechanisms involved in leg sensory organ patterning, a Hedgehog (Hh)- and Decapentaplegic
(Dpp)-responsive enhancer of the h gene, which directs expression of
h in a narrow stripe in the dorsal leg imaginal disc (the
D-h stripe) has been examined. These studies suggest that the domain of D-h
expression is defined by the overlap of Hh and high-level Dpp signaling. The D-h enhancer consists of a Hh-responsive activation
element (HHRE) and a repression element (REPE), which responds to the
transcriptional repressor Brinker (Brk). The HHRE directs expression of
h in a broad stripe along the anteroposterior (AP) compartment
boundary. HHRE-directed expression is refined along the AP and dorsoventral
axes by Brk1, acting through the REPE. In D-h-expressing cells, Dpp
signaling is required to block Brk-mediated repression. This study elucidates
a molecular mechanism for integration of the Hh and Dpp signals, and
identifies a novel function for Brk as a repressor of Hh-target genes (Kwon, 2004).
The D-h and V-h
stripes are regulated by separate enhancers, which map between 32-38 kb 3' to the h transcription unit.
ac stripes are not expressed until 6 hours after pup